The overall goal of this research is to investigate basic mechanisms of calcification in endochondral bone. Specifically, we will test hypothesis concerning the function of matrix vesicles in cartilage mineralization, utilizing a new technology for modifying these vesicles byfusing them with synthetic phospholipid liposomes. These hybrid or "recombinant" vesicles are useful reagents for the study of matrix vesicle function, as they can be constructed to contain a defined intravesicular medium and an enhanced membrane content of selected proteins including alkaline phosphatase, an enzyme long considered to play an important role in mineral metabolism. One aim of this proposal is to construct hybrid vesicles which differ in membrane content of acidic and neutral phospholipids, and to assess the ability of these hybrid membranes to take up Ca2+, PO4 and to mineralize in vitro. Further, we will construct recombinant vesicles which differ in intravesicular pH or ion content, and we will ascertain whether these imposed ion and pH gradients can facilitate the initiation of mineral formation by the vesicles. Finally, we will construct hybrid vesicles enriched in alkaline phosphatase, proteolipid and calcium-phospholipid-phosphate complex, all of which have been shown to enhance mineralization in vitro. The results of these studies will permit critical evaluation of long held theories of matrix vesicle action, and are likely to lead to a new understanding of the role of these enigmatic particles in the formation of hard tissues.